Filamentous fungi are known for their ability to produce a wide range of different metabolites and, in particular, secreted enzymes with various industrial applications including the provision of therapeutics or for use in the bakery industry.
Prior to development of fungal transformation systems, improvement of production strains was largely restricted to a strategy based on classical mutagenesis followed by screening for a phenotype or enzymatic activity desired and selection of the best producer strain.
Commercially, filamentous fungi have now become widely used host cell systems for the production of both homologous and heterologous proteins and to date, several molecular and genetic approaches have been successfully applied to optimise filamentous fungi, in particular those from the genus Aspergilli, as a cell factory.
In many cases, these approaches have led to elevated yields of both homologous and heterologous proteins, and substantially reduced the time required for implementation of a new production process at a large scale.
However, there remains a need for improved methods.
Filamentous fungi are well-known producers of cellulolytic and hemicellulolytic enzymes. The cellulase degradation system of these organisms consists of three classes of enzymes, endoglucanases, cellobiohydrolases and beta-glucosidases. Xylans are hemicellulose, heterogenous polymers, which require a more complex set of enzymes for their degradation. These enzymes, broadly classed as xylanases, include endoxylanase, beta-xylosidase, acetylxylan esterase, α-L-arabinofuranosidase, arabinoxylan arabinofuranohydrolase, beta glucuronidase, feruloyl esterase and p-coumaroyl esterase (Scheme 1).
The expression of cellulose- and xylan-degrading enzymes by Aspergillus and Trichoderma species is regulated at the transcriptional level. However, the mechanisms of repression and induction have not yet been completely elucidated.
The transcription factor xlnR was originally cloned from the filamentous fungus A. niger and identified as a transcriptional activator of xylanase-encoding genes (WO 97/00962). In fact, it has been shown to trigger the transcription of a broad set of genes involved in degradation of cellulose and hemicelluloses in a number of fungal species. Based on its structure, XlnR has been ascribed to a family of fungal transcription factors, whose DNA-binding domain is composed of 6 conserved cysteine residues chelating two Zn atoms, a so-called Zn binuclear cluster.
xlnR homologues have been cloned from various Aspergilli (A. nidulans, A. oryzae, A. kawachii, A. tubingensis, A. flavus), as well as from other fungi, like Hepocrea jecorina (anamorph: T. reesei). Moreover, similar sequences can be found in many publicly accessible fungal genome databases, such as Aspergillus fumigatus, Neurospora crassa, Magnaporthe grisea, Podospora anserina, Fusarium grameniarum. Though the overall homology between different species varies in the range of 50-90%, it exhibits nearly 100% identity within the DNA binding domain encompassing a cluster of six cysteine residues at the N-terminal part of the protein. In particular, a stretch of aa residues between the second and third cysteines represented by the sequence NQLRTK (SEQ ID NO: 7) is extremely conserved. In general, this sequence determines the DNA-binding specificity of a Zn2Cys6 binuclear cluster protein, which, in the case of XlnR, recognises a cognate DNA target with the consensus core 5′ GGCTAR 3′ (de Vries and Visser, 2001; Microbiol Mol Biol Reviews v65: 497-522). Therefore, all these homologues could be predicted to play a role in transcriptional regulation of genes related to xylan degradation.
Lately, several approaches have been made to improve the production of proteins of interest (POIs) using recombinant host cells from filamentous fungi.
The most straightforward strategy for improving the yield of secreted homologous and heterologous proteins documented in the literature is to introduce multiple copies of a structural gene of interest (see, for example, Hessing et al, 1994). However, this may not always lead to overexpression of the gene of interest. The concentration of a protein of interest in the fungal cell has also been increased by expressing it from a strong promoter (Mathieu and Felenbok, 1994; Nikolaev et al., 2002; Rose and van Zyl, 2002).
Transcription of the xylanolytic and cellulolytic system and, as a consequence, the total enzyme production by Aspergillus niger could be enhanced by increasing the gene copy number of xlnR (Gielkens et al., 1999). Conversely, inactivation of xlnR by gene disruption leads to the loss of transcription of extracellular xylanolytic genes.
Another approach implies a modification of the promoter region of the gene of interest by mutating binding sites of a transcription factor. Promoter activity of the Aspergillus oryzae xynF1 gene, monitored by β-galactosidase activity, was successfully upregulated by mutating two non-canonical XlnR binding sites to a sequence, which is supposed to have the highest binding affinity (Marui et al., 2003). Transformants carrying three canonical XlnR binding sequences in the xynF1 promoter region produced 2.8 times more enzyme than those with the authentic promoter.
Many of the genes involved in carbon metabolism are subject to carbon repression mediated by the global repressor CreA. In fact, CreA controls gene expression at several levels and can repress both a structural and regulatory gene. Deletion or mutation of the CreA binding sites in the corresponding promoters results in partial derepression of transcription and improved protein production (Orejas et al., 1999). A further improvement was obtained in a creA derepressed background (Prathumpai et al., 2004).
In Aspergilli, D-xylose acts as the inducer of xylanolytic genes (de Vries et al, 1999; Gouka et al, 1996). In addition, xylanase production appeared to be improved in both T. reesei (Xiong et al, 2004) and Penicillium canescens (Vavilova et al., 2003) grown on L-arabinose-containing media.
Metabolic control analysis and metabolic pathway engineering are other helpful means of strain improvement. It has been shown that the flux control of xylose metabolism is exerted at the first two steps (Prathumpai et al., 2003). Disruption of one of the genes responsible for D-xylose reduction resulted in an increase of xylanase transcription (Hasper et al., 2000).
The most spectacular improvement of heterologous protein production yields was obtained by the C-terminal fusion of a protein of interest to a well-secreted fungal protein (Ward et al., 1990, Contreras et al., 1991).
In addition, deletion of genes with non-desirable activities, like proteases, appears to be extremely useful to elevate expression (Van den Hombergh et al., 1997).
It is an object of the present invention to provide improved methods for increasing production of proteins of interest in host cells in which the activity of a transcription factor involved in metabolic regulation has been modified.